Molecular Analysis of Xq28-Linked Incontinentia Pigmenti

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DESCRIPTION (provided by applicant): Incontinentia pigmenti (IP) is an X-linked dominant and male-lethal disorder that mainly affects the skin, teeth, eyes, and central nervous system. Females survive because of selective proliferation of cells expressing the normal X chromosome and therefore, skewing of X-inactivation in females is a hallmark feature of IP. We recently identified the gene involved in IP and demonstrated mutations in multiple IP patients. The gene, NEMO, is involved in the NF-kB signal transduction pathway and its absence renders cells susceptible to apoptosis. Hence, loss-of-function mutations in males result in embryonic lethality in humans as well as mice. While knockout models of Nemo demonstrate a classic IP phenotype in female mice, the males die during embryonic development. Female IP patients demonstrate a phenotype presumably because of the X-inactivation pattern and the timing of apoptosis in cells expressing the mutant X chromosome. A majority of IP patients carry a deletion of exons 4-10 that occurs due to misalignment, possibly during recombination, of repeats located in intron 3 and at the end of the gene. The remaining patients have demonstrated microdeletions, duplications, and substitutions. Nearly all of our patients exhibit loss-of-function mutations. Although IP has been considered a classic male-lethal disorder, we recently identified mutations in males also, which appear to be hypomorphic and not lethal to the cellular environment. When males have IP, they present other signs not typically associated with IP. Therefore, numerous male IP cases may have been mistaken for another similar disorder due to previous misconceptions about male lethality in classic IP and the presence of atypical signs in surviving IP males. Hence, one of the goals of this project is to examine patients with similar disorders to see if they are allelic with IP. Variant phenotypes due to hypomorphic mutations are likely to reveal significant information about the functioning of NEMO and the NF-kB pathway. In this respect, we have proposed the creation of transgenic mice expressing nonlethal, hypomorphic mutations to investigate the various functions of the NF-kB pathway and the pathogenesis of IP. These transgenic mice will help address the hypothesis that hypomorphic mutations lead to an unstable protein with residual activity and that the specific timing of expression of the mutation leads to the phenotypes observed in humans. As a third goal, this project focuses on understanding NEMO expression by analyzing its product. This effort is expected to provide insight into the control of NEMO expression and its consequence on downstream activity via the NF-kB pathway. Lastly, mutation detection in IP patients is complicated by the presence of a second copy of NEMO located ~75kb distal to the first copy. These two copies share nearly 100% homology between exon 3 and ~10kb distal to exon 10. The second copy (NEMO) lacks exons 1 and 2 and may not be functional. The fourth goal of this project is, therefore, to determine role of this repeated copy in IP mutation. Collectively, the aims of this project will help understand the pathogenesis of IP in males, the regulation of the NF-kB signaling pathway, the regulation of NEMO expression, and the nature of the two NEMO copies.